1. Field of the Invention
This invention relates to a method for using solid phase extraction materials to purify nucleic acid samples contaminated with proteinaceous material.
2. Background
T. Maniatis, et al. [Molecular Cloning - A Laboratory Manual, (New York: Cold Spring Harbor Laboratory, 1982), pp 458-460] describe a method of purifying nucleic acids based on the procedures described by K. S. Kirby [Biochim. J., Vol. 66, 494-504 (1957)] and J. Marmur [J. Mol. Biol , Vol. 3, 208-218 (1961)]. This method uses phenol in a liquid/liquid extraction procedure, whereby the protein contaminating an aqueous nucleic acid sample is extracted into the phenol phase, leaving the nucleic acid in the aqueous phase. This nucleic acid purification procedure may actually involve a series of liquid/liquid extractions, in which an aqueous volume of nucleic acid sample is sequentially extracted with equal volumes of liquified phenol, phenol/chloroform/isoamyl alcohol (50/48/2), and chloroform/isoamyl alcohol (96/4). After each extraction, the organic phase containing protein (and some nucleic acid) is back-extracted with an aqueous buffer to recover any nucleic acid extracted into the organic phase. The aqueous nucleic acid sample must then be exhaustively extracted with diethyl ether to remove any residual phenol, chloroform, or isoamyl alcohol. Finally, the residual diethyl ether is removed either under reduced pressure or by blowing nitrogen over the sample for ten minutes. Removal of the residual organic solvents is of paramount importance, as they would otherwise interfere in subsequent cloning or hybridization procedures in which the nucleic acid might be used.
Although phenol extraction is a very effective method for the purification of nucleic acid samples and is very widely used, the procedure is also labor-intensive and time-consuming. Other disadvantages of this technique include the hazards (chemical irritant, carcinogen, stench and flammability) associated with the use of the organic solvents (phenol, chloroform, isoamyl alcohol and diethyl ether).
A second method of nucleic acid purification is based on the preferential adsorption of nucleic acids using NENSORB 20.TM. Nucleic Acid Purification Cartridges (DuPont). A proteinaceous DNA sample is passed through a column of NENSORB.sup..TM. particles, binding the DNA to the particles, and allowing most of the proteinaceous material to be washed from the column with an aqueous buffer. The bound nucleic acid is then desorbed from the particles by passing 20% aqueous ethyl alcohol (or 50% aqueous methyl alcohol) through the column and the DNA is collected in the column effluent Prior to use of the DNA in cloning or hybridization, the alcohol must be removed from the DNA solution by pulling a vacuum on the sample for several hours.
The use of NENSORB.TM. has other limitations The NENSORB.TM. material, besides binding nucleic acid, also binds protein to a variable extent This not only limits the capacity for quantitatively binding nucleic acid from a sample heavily contaminated with proteinaceous material (e.g., blood serum, tissue homogenate), it also affords the possibility that some loosely bound protein may elute with the nucleic acid when the latter is desorbed from the NENSORB.TM. by the use of aqueous alcohol. Three different solvents are needed to activate the NENSORB.TM. column, wash the unbound protein from the column, and then elute the nucleic acid from the column. Nucleic acid is eluted from the column in a solvent (20% ethyl alcohol or 50% methyl alcohol) that is incompatible in protocols of subsequent uses of the nucleic acid (e.g., cloning, hybridization assays, etc.). The purification procedure using NENSORB.TM. can be time-consuming, requiring an hour or more to purify a single sample of nucleic acid.
C. A. Thomas et al., Analytical Biochemistry, Vol. 93, 158-166 (1979), disclose the use of glass fiber filters to adsorb protein and protein-DNA complexes from aqueous DNA samples. This method suffers from the following disadvantages: (1) The aqueous sample of DNA must contain at least 300 mM NaCl for the protein to bind effectively to the glass fiber filter. This relatively high salt concentration would have to be lowered by removing the salt from the DNA sample (or diluting the solution) prior to further use of the DNA in biological reactions, such as digesting the DNA with a restriction enzyme. (2) The recovery of DNA from the filter is high only if the filter is extensively washed with an aqueous buffer. (3) The low specific surface area (approx. 2 m.sup.2 /g) of the glass fibers results in a low protein-binding capacity of the glass fiber filter. The maximum protein-binding capacity is estimated to be 8.5 mg of bovine serum albumin (B$A) per gram of glass fiber filter.
B. Vogelstein et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 26, No. 2, 615-619 (1979), disclose a method for separating DNA from agarose by binding the DNA to glass, preferably glass powder, in the presence of NaI. The DNA was removed from the glass by elution with Tris.multidot.HCl, pH7.2/0.2M NaCl/2 mM EDTA. It is also disclosed that silica gel and porous glass beads are unsuitable for DNA purification.
J. Kirkland, J. Chromatographic Sci., Vol. 9, 206-214 (1971), discloses the preparation of surface-modified silica gels by reacting silane reagents with the surface of the porous shell of Zipax.TM. controlled-surface chromatographic support.
J. Kohler, et al., J. Chromatography, Vol. 385, 125-150 l(1987), disclose the preparation of fully hydroxylated calcined silica by the dissolution and redeposition of silicic acid.
T. Watanabe et al., J. Solid-Phase Biochem., Vol. 3, 161-173 (1978), discloses the preparation of immobilized tannins for protein adsorption.
The object of this invention is to provide an improved solid-phase extraction procedure for removing proteinaceous contaminants for nucleic acid samples. The method should be capable of removing large proportions of proteins from minute quantities of nucleic acid and recovering the latter in a biologically active state. The method should also be rapid, convenient, provide quantitative recovery of the nucleic acid and minimize the use of hazardous materials. It should also not introduce contaminants or impurities into the nucleic acid sample that may interfere in subsequent uses of the nucleic acid.